Pituitary transplantation is an effective treatment for hypopituitarism but lack of pituitary cells with secretory function has slowed down its progress. The present study explores changes to hormone production and morphology of murine pituitary cells in pseudo-microgravity culture. It aims to improve current concepts of culture methods for pituitary cells that may provide new options for pituitary transplantation in the future. The RCCS-4H simulated microgravity 3D method was used for primary pituitary cell culture. Primary pituitary cells were isolated and cultivated in both 2D and 3D cell culture. Cell morphology was observed by visual light and immunofluorescence microscopy. Hormone production was measured by ELISA. Pituitary cells that were cultured in the 3D pseudo-microgravity system maintained their natural morphology and function. Growth rate and hormone secretion of 3D-cultured pituitary cells were higher from day 5 to day 17 as compared to that of 2D cell culture. These results indicate that pseudo-microgravity culture facilitates murine pituitary cells to maintain a natural morphology, better survival time, and physiological hormone production. This method therefore might be suitable for the cultivation of analogous human pituitary cells that may serve as excellent donor cells for cell transplantation therapy in patients with hypopituitarism.